Lipid peroxidation in tissue and in tissue fractions represents a degradative process, which is the consequence of the production and the propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids, and has been implicated in the pathogenesis of numerous diseases, including systemic lupus erythematosus (SLE). We have found that bovine serum albumin incubated with peroxidized polyunsaturated fatty acids significantly cross-reacted with the sera from MRL-lpr mice, a representative murine model of SLE. To identify the active substances responsible for the generation of autoantigenic epitopes recognized by the SLE sera, we performed the activity-guiding separation of a principal source from 13-hydroperoxy-9Z,11E-octadecadienoic acid and identified 4-oxo-2-nonenal (ONE), a highly reactive aldehyde originating from the peroxidation of ω6 polyunsaturated fatty acids, as the source of the autoantigenic epitopes. When the age-dependent change in the antibody titer against the ONE-modified protein was measured in the sera from MRL-lpr mice and control MRL-MpJ mice, all of the MRL-lpr mice developed an anti-ONE titer, which was comparable with the anti-DNA titer. Strikingly, a subset of the anti-DNA monoclonal antibodies generated from the SLE mice showing recognition specificity toward DNA cross-reacted with the ONE-specific epitopes. Furthermore, these dual-specific antibodies rapidly bound and internalized into living cells. These findings raised the possibility that the enhanced lipid peroxidation followed by the generation of ONE may be involved in the pathogenesis of autoimmune disorders.